Motility scanner and method

ABSTRACT

An improved motility scanner is disclosed for characterizing the motion of sperm, bacteria, particles suspended in flowing fluids and the like. The motility scanner includes an improved optical system, a source of illumination for the system and radiation sensing means including electronic image reversal means coupled to the system. A disposable specimen holder allows external loading thereof and its positioning on a heated specimen support. The illumination source functions as a collimator in directing all of the emanating illumination onto the specimen. Both directly transmitted light and light scattered by the specimen are received by an imaging lens and are both focussed thereby onto pixels of a light sensitive device. A movable plate provided with a retarding member is designed to be located at the plane conjugate to the plane of the small source aperature. The directly transmitted light is transmitted through the retarding member and/or the attenuating member. The light scattered by the specimen, however, travels for the most part through the plate in areas not covered by the retarding and/or attenuating members.

This is a continuation-in-part of pending application Ser. No. 897,036filed Aug. 15, 1986, entitled "Motility Scanner and Method."

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the characterization of themotion of particles suspended in a liquid medium and, more particularly,to an improved device and a method developed for characterizing themotion of sperm, bacteria, particles suspended in flowing fluids,Brownian motion and the like.

2. The Prior Art

The characterization of the motion of particles suspended in a liquidmedium is of particular significance in fertility analysis. The term"characterization" as used in this specification and in the appendedclaims is intended to define a procedure involving analyzing anddetermining the motion of particles in a liquid medium. Motilityanalysis as regards fertility includes the determination of spermmotility and mean sperm velocity. The term "sperm motility" is intendedto to be defined as the fraction of sperm moving among all the sperms ina given specimen sample. The term "progressive motility" is intended todefine the fraction of sperm moving in an approximately constantdirection. The term "progressivity" or "linearity" is the ratio betweenthe distance travelled and the track length.

Motility analysis is undertaken regularly for animals, such as horses,in particular race horses, and prime bulls, in order to establish and tokeep a permanent running record of the quality of their semen, hencetheir breeding potential. Motility analysis also represents an importantsegment in diagnosing certain reproductive problems in the human male.

For the most part, sperm motility and mean sperm velocity are simplyestimated by visual examination of a drop of semen on a slide. Theresults of such visual examinations vary widely, often by as much as40%, from one observer to another. Further, one cannot estimate, purelyon a visual examination, linearity or velocity distribution functions.In order to determine such linearity or velocity distribution functions,a method of multiple exposure time-lapse photography has been developed.This method is tedious and time consuming in that it requires the manualcounting of the sperm tracks, followed by manual derivation of thedistributions of linearity and velocity. In order to speed up thismanual method, a computerized version thereof has been developed whichallows for the calculation of the distribution functions, but only afterthe sperm tracks first have been manually outlined by using aninteractive indicating device such as a light pen or a "mouse". Afurther improved version employs a microscope attached to a computer,video recorder and other peripheral items. This improved version isdesigned to analyze a drop of semen in a special cell, called the Maklercell. The Makler cell is such that it maintains an exact narrow spacing,usually 0.01 mm, between its opposed walls. Such an exact narrow spacingis required so as to provide a sharp focus for the microscope image, toreduce the visual density and thus enable the computation of spermdensity. The narrow spacing of the Makler cell, however, constricts themotion of the sperm tails. Sperm tails are believed to require 0.02 to0.10 mm diameter about their axes, depending on species, in order tooperate and move freely, i.e., without constriction. Consequently, asystem employing the narrow Makler-type cell spacing adversely affectsthe very quantities, e.g., motility, velocity and linearity, that it isdesigned to measure. Such system also makes it very difficult to measurethe motion of sperm in a diluent since sperm density is reduced to sucha degree that obtaining statistically significant numbers of spermrequires a long time, and the superposition of many successive fields.The measurement of sperm motion in a diluent is frequently required inmotility analysis. For example, in in vitro fertilization experiments,the semen may be diluted by a factor of 100 to 500. Further, the Maklercell is expensive to make and, in use, is difficult to maintain at anexact, controllable temperature.

In said prior pending application Ser. No. 897,036 filed Aug. 15, 1986,we have disclosed a motility scanner in which only light scattered byand from the specimen is able to reach the optical system. Morespecifically, the design of the prior motility scanner is such as toassure that the imaging lens thereof receives no directly transmittedlight. As a result, the image formed appears bright on a darkbackground, a dark field image. Two significant advantages derivetherefrom. First, analysis of sperm motion is facilitated since only theimportant objects, the sperms, appear bright and the microprocessorsystem is easily programmed to consider only bright objects. Second, aconsiderable depth of focus can be obtained, allowing thereby the use ofspecimen holding chambers of a size in which the motion of the spermcells is not constrained by the presence of walls. These advantages,albeit remaining significant, are nevertheless obtained at the cost ofpoorer image resolution. Consequently, sperm cell heads appear asextended spots, and the sperm cell tails are visible only, if at all, inrare circumstances. As a result, it is impossible to tell what portionof the non-moving objects represents detritus or non-motile sperm cells.In accordance with the present invention, the presence of a visiblesperm cell tail resolves this problem. A sharper image resolution alsocauses the sperm cell heads to appear reduced in size of the total areacovered by the sperm cell heads. This reduced area, in turn, decreasesthe probability of sperm cell head collisions, since the apparentcross-section is smaller. The proportion of tracking errors at a givensperm cell concentration is markedly reduced thereby. Further, accurateanalysis at higher values of sperm cell concentrations also is nowpossible.

SUMMARY OF THE INVENTION

It is a principal object of the present invention to overcome the abovedisadvantages by providing an improved motility scanner forcharacterizing the motion of sperm, bacteria, particles suspended inflowing fluids and the like.

More specifically, it is an object of the present invention to providean improved motility scanner for characterizing the motion of sperm,bacteria, particles suspended in flowing fluids and the like, comprisingan improved optical system including an imaging lens; a source ofillumination provided for the optical system; a heated specimen supportdisposed in the object plane of the imaging lens; a specimen holder,preferably disposable, designed to be positioned on the heated specimensupport; radiation sensing means coupled to the optical system at theplane conjugate to the plane of the specimen; signal processing means,including electronic image reversal means, for analyzing the signalsgenerated by the radiation sensing means; and display means connected tothe signal processing means for displaying information characteristic ofthe motion of the sperm, bacteria, particles suspended in flowing fluidsor the like. Preferably, the source of illumination comprises a singleLED, combined with a small source aperture, functioning as a collimatordirecting its illumination onto the specimen in the specimen holder,with both the directly transmitted light and the light scattered by thespecimen being admitted into the imaging lens. A movable plate, providedwith a centrally disposed retarding member and/or attenuating member, isdesigned removably to be located in the beam of radiation of the imaginglens at a plane conjugate to the plane of the small source aperture.With the movable plate in the path of the beam, the directly transmittedlight is transmitted through the retarding and/or attenuating member.The light scattered by the specimen however travels, for the most part,through the plate in areas not covered by the retarding and/orattenuating members. Preferably, the motility scanner also is containedwithin a portable or transportable housing. The motility scanner isprovided with a transformer and a power cord whereby it can be connectedto a conventional 115 V.A.C. power source. The improved motility scannercan be used in one of three operative modes: (a) with bright fieldillumination; (b) with dark field illumination; or (c) with phasecontrast in dark field illumination.

Other objects of the present invention will in part be obvious and willin part appear hereinafter.

The invention accordingly comprises the motility scanner of the presentdisclosure, its components, parts and their interrelationships, thescope of which will be indicated in the appended claims.

Brief Description of the Drawings

For a fuller understanding of the nature and objects of the presentinvention, reference is to be made to the following detaileddescription, which is to be taken in connection with the accompanyingdrawings, wherein:

FIG. 1 is a schematic elevation, partly in section, of a motilityscanner constructed in accordance with the invention;

FIG. 2 is an isometric perspective view, on an enlarged scale, of a partof the motility scanner shown in FIG. 1;

FIG. 3 is a plan view, along the line 3--3, of a portion of the motilityscanner of FIG. 1;

FIG. 4 is a view, on an enlarged sale and along the line 4--4 of anotherpart of the motility scanner shown in FIG. 1;

FIG. 5 is a plan view of an alternate specimen holder;

FIG. 6 is a plan view of still another specimen holder;

FIG. 7 is a partial view, similar to FIG. 1, but showing anotherembodiment of the motility scanner;

FIG. 8 is a plan view along the line 8--8 of FIG. 7;

FIG. 9 is a partial view, similar to FIG. 1, but showing an improvedoptical system for the motility scanner of the present invention; and

FIGS. 10 through 13 illustrate a part of the improved optical system ofFIG. 9.

Detailed Description of the Preferred Embodiment

In general, the illustrated embodiment of a motility scanner 10, asdepicted in schematic elevation and partly in section, is designed forthe characterization of the motion of particles suspended in a liquidmedium and has particular application to fertility analysis. In additionto motility analysis of sperm, the motility scanner 10 also is useful inanalyzing and determining the motion of other items of interest, such asbacteria, particles suspended in flowing fluids, Browninan motion, andthe like.

The motility scanner 10, as illustrated in FIG. 1, essentially comprisesan optical system 12, a source of illumination 14 for the optical system12, a heated specimen support 16, radiation sensing means 18 coupled tothe optical system 12, signal processing means 20 coupled to theradiation sensing means 18, display means 22 coupled to the signalprocessing means 20, and control means 24 for operating the motilityscanner 10. Preferably, the motility scanner 10 is mounted within aportable housing 26, including a top wall 28, a bottom wall 30, and fourside walls, two of which 32 and 34 are shown in FIG. 1. In theembodiment of FIGS. 7 and 8, the source of illumination comprises one ormore LED's, as hereinbelow more fully described.

Preferably, the display means 22 and the control means 24 are located ona panel 36 set into the front side wall, not shown. The motility scanner10 also preferably includes a power supply 40 and a power cord 42,having a plug 44, connected thereto. The source of illumination 14preferably is connected in parallel to both the battery pack 38 and thetransformer 40. It is to be understood that whenever the plug 44 isplugged into a conventional power outlet of 115 V.A.C., the internalbattery pack 38 is automatically disconnected from also supplying powerto the illumination source 14 and will remain so disconnected as long asthe plug 44 remains plugged into the conventional power outlet. Aconvenient handle 46 is secured to the top wall 28, centrally thereof.

A specimen to be characterized by the motility scanner 10 is externallyloaded into a specimen holder 50, which is then placed on a motorizedand heated specimen support 16. The heated specimen support 16 slidesthrough a slot 52 formed in the side wall 32, thereby retracting toposition the specimen at the imaging point.

The Optical System of FIG. 1

The optical system 12 of the motility scanner 10 comprises a collimatinglens 56, a condensing lens 62 and an imaging lens 64. A pair ofreflecting elements 58 and 60 may be included to fold the light path.Preferably, the reflecting element 58 comprises a fully reflectingprism, as shown, and the reflecting element 60 comprises a front surfacemirror. If desired, the element 58 also can be a mirror, or the like. Itwill be noted that the specimen support 16 and, more precisely, thethereon disposed specimen holder 50, are mounted at or near the focalplane of the condensing lens 62.

A focussing means 120 is operatively coupled to the imaging lens 64 toallow for the variation in the distance between the lens 64 and thespecimen support 16. Also preferably one or more optical filters 119 arepositioned between the collimating lens 56 and the reflecting element58. These filters 119 are intended to remove unwanted spectral portionsof the radiation emitted by the source 14.

The specimen holder 50 also is positioned during analysis at the objectplane of the imaging lens 64, with the radiation sensing means 18 beingdisposed at the imaging plane of the imaging lens 64. An obscuringmember 66, which preferably is a disk, is positioned on the exit side ofthe prism 58. It is to be understood that, in the alternative, theobscuring member 66 also can be bonded directly to the exit or entrysides of the prism 58. The function of the obscuring member 66, incooperation with an aperture 68, is to assure that the imaging lens 64receives no directly transmitted light. In the preferred embodiment, thecondensing lens 62 produces an image of the obscuring member 66 on theaperture 68. Therefore, the imaging lens 64 accepts only light scatteredor refracted into a cone of acceptance 70 by the specimen containedwithin the specimen holder 50. Consequently, any object present in thespecimen which scatters or refracts light, such as a spermatozoon, willappear bright on a black background.

The imaging lens 64 is a single compound lens having an effective depthof field at its object plane of at least about 0.2 mm, and preferablyabout 0.5 mm, the significance of which will become apparent below. Thefocal length of the imaging lens can vary between about 2.5 cm to about5 cm, with the latter being preferred. The magnification ratio of theimaging lens 64 preferably is less than ten-to-one and, typically isabout four-to-one. The imaging lens 64, for example, can be a "SchneiderComponon" enlarger lens made by the Schneider Kreuznach company inGermany.

The Radiation Sensing Means

The radiation sensing means 18 of the motility scanner 10 preferablyincludes a light-sensitive device 76 provided with a plurality oflight-sensitive cells 78, known as pixels, observe FIG. 4. The device76, preferably a charge coupled device (CCD) or the like, generates ananalog signal representative of the focused image on the pixels 78a ofthe device 78. Each pixel 78a is about 0.01 mm to about 0.02 mm inwidth. Since a typical sperm cell head has a diameter from about 0.002mm to about 0.005 mm, one sperm cell head can be imaged on severalpixels, using magnification 4 to 10. Further, each pixel 78a is designedso as to be able to discriminate, with uniform and stable sensitivity,up to sixty-four distinct levels of optical illumination intensity.Thus, light-scattering particles found in the specimen underinvestigation, such as spermatozoa, will appear bright in a dark field.

The analog signal representative of the focused image on the pixels 78is transmitted via lead 77 to an analog to digital converter 80, alsoknown as a frame grabber board, where the signal is converted to digitalform on lead 79, and stored in RAM, preferably a 64 KByte RAM, fromwhich it is available to the signal processing means 20. The signalprocessing means 20 essentially comprises a microprocessor 82 and apre-programmed device 84. The microprocessor 82 preferably is capable ofaccessing in excess of a 1.5 MByte RAM included in the system, while thedevice 84 contains a plurality of pre-programmed read-only memory (ROM)chips. The output of the signal processing means 20 is transmitted vialead 81 to the display means 22. Display means 22 basically includes ascreen 22a and a printer 22b. The screen 22a is a typical CRT. In oneform it is black and white. In other forms it can displaycolorcombinations and/or information in both graphical and alphanumericform. The printer 22b preferably is a hard copy printer designed toreproduce the information appearing on the screen 22a on paper uponcommand of the operator, effected by depressing one of the controlbuttons of the control means 24. In one embodiment, the printer is anintegral part of the motility scanner. In another embodiment it is anexternal peripheral. A power switch 88 allows the motility scanner 10 tobe rendered operational.

The Specimen Holder

The specimen holder 50 for the motility scanner 10 is shown, inisometric perspective and on an enlarged scale, in FIG. 2, and itsoperative use is illustrated in FIG. 3. As noted, the specimen holder 50comprises a flat tubular member 90 formed of a hard, transparentmaterial, such as glass or plastic, with a pair of adjacently spacedwide parallel sides 92 and 94 and remotely spaced short, curvedconnecting sides 96 and 98, cooperately defining therebetween a specimenholding channel 100. Preferably, the specimen holding channel 100, whichis open at its respective ends, is formed with a depth 102 of about 0.2mm between its wide parallel sides 92 and 94, a width 104 of about 2 mmbetween its curved connecting sides 96 and 98, and an axial length 106of about 5 cm between its respective open ends.

The operative use of the specimen holder 50 is best described withreference to FIGS. 1 and 3. After the specimen holding channel 100 ofthe specimen holder 50 has been filled with a properly diluted sample,as more fully described below, the specimen holder 50 is placed on theretractable heated specimen support 16, which slides through the slot 52in the side wall 32. Specimen support 16 includes a flat member 110,provided with a window 112 and a longitudinal slot 114. Member 110 isdesigned to slide in and out of the slot 52, as indicated by an arrow115, with the aid of means 117. Means 117 preferably is a motorizedmeans and slides with the aid of a small electric motor 113. Member 110preferably is heated via an electrical resistance wire 116 embeddedtherein. Member 110 also is provided with a further and wider slot 124designed to accomodate a microscope slide 125, if that is chosen tocontain the specimen. In addition, member 110 also has a circular slot126 designed to accomodate a Makler-type cell 127, if that is chosen tocontain the specimen. It should be noted that each of these slots 114,124 and 16 is so located as to center the respective specimen holderover the window 112.

When the microscope slide 125 is used to hold the specimen, the densityof the sperm population therein is not known since the spacing betweenthe slide 125 and its coverslip is not known. A user of the motilityscanner 10 would find the employment of the Makler-type cell 127particularly useful whenever more concentrated sperm dilutions were tobe examined.

It should also be noted that the source of illumination 14 is completelyenveloped, save for an opening 49 for the collimating lens 56, and anair cooling aperture 51, in a suitable enclosure 48.

The Embodiment of FIGS. 7 and 8

In the embodiment illustrated in FIGS. 7 and 8, a simplified opticalsystem 130 is employed in the motility scanner due to a different sourceof illumination 132 used. This source of illumination 132 comprises atleast one, and preferably more LED's 134, mounted in a ring-like mannerin a support member 136. The support member 136 is provided with aplurality of apertures 138 to accomodate the LED's 134. Each of theseapertures 138 is formed at an angle 142, inclined with respect to acentral axis 140 of the member 136. The LED's are then mounted in theapertures 138 at this inclined angle 142, which is the angle between themaximum emission of radiation from the LED's 134 and the axis 140. Thisangle of convergence 142 can range from about 15° to about 45°, andpreferably is about 25°. Variation in the angle of convergence 142 willeither enhance or or weaken the motility scanner's ability to detectsperm rotation within the specimen holder 50. This is so since spermrotation causes the sperm cell's image to increase or to decrease inintensity. The amplitude of this intensity change is related to theangle of incidence of the illumination on the sperm cell within thespecimen holder 50.

Each LED 134 produces an intense, concentrated beam of forward directedradiation 144 directed at a point 146 of intersection of the specimenholder 50 and the central axis 140. Since each LED 134 independentlyilluminates the whole field of investigative interest of the specimenholder 50, one such LED 134 can suffice. In order to enhance theuniformity of illumination at the specimen holder 50, however, it ispreferred that a ring of LED's 134 be employed. The number of LED's inthe ring can vary anywhere from three to about twelve, with a ring ofsix LED's being preferred, as illustrated in FIG. 8. The geometry of thering of LED's 134 is so designed as to prevent direct radiation from theLED's 134 from reaching and entering the imaging lens 64. Thus, the onlyradiation to reach and enter the imaging lens 64 is radiation within acone of acceptance 148, which is radiation scattered by the particleswithin the specimen holder 50 whose motion the motility scanner isdesigned to characterize. These particles will, therefore, appear brightin a dark background.

Although any type of LED's can be used in the structure described withreference to FIG. 7, LED's emitting radiation in the infrared region arepreferred. This is so since radiation in the infrared range, andparticularly about and near 880 nanometers, propagates well through theoptical glass frequently employed for making the specimen holder 50 andthe optical train. Further, the sensitivity of the CCD detector 76 atand near 880 nm is high, yielding a clear image, with adequatebrightness. Still further, by using the longer wavelengths ofilluminating radiation about and near 880 nm, we find that these longerwavelengths are less subject to scattering by smaller particlessuspended in the diluent medium, whose presence otherwise mightadversely affect the output results of the motility scanner. Forexample, when using a typical milk-based diluent medium for spermanalysis, one of the problems frequently encountered consists in themilk particles obscuring the optical transmission through the specimenholder 50 and in increasing the amount of scattering by such milkparticles. As a consequence, the definition with which sperm cellsnormally can be discerned is markedly reduced. By using IR radiation,the scattering from the sperm tails also is reduced, enhancing therebythe characterization of sperm head movement. Unwanted scattering ofinfrared radiation, when compared to visible light, is greatly reduced,and consequently the image quality obtained is improved. At present, weprefer to use IR radiation emitting LED's, with an intensity half angleof 8° and having a peak emission at or near 883 nm, such as the GaAlAsLED's. Further, we prefer to operate these LED's at about 50% to about75% power levels only so as to markedly increase their operationallifetimes.

It will be observed that in the structure of FIGS. 7 and 8 employing theLED's 134, as above described, in addition to the enhanced opticalefficiency gained at a reduced generated heat (only about 2 watts heatas contrasted with about six times as much heat generated by aconventional hot filament lamp), both the collimating lens 56 and thecondensing lens 62 are omitted, together with the prism 58 and thethereon placed obscuring member 66.

The Embodiment of FIGS. 9-13

In the embodiment illustrated in FIGS. 9-13, an improved optical system160 is employed in the motility scanner of the invention. The improvedoptical system 160 excels at increased image resolution. As aconsequence, non-moving objects appearing in the specimen underinvestigation can now be differentiated as non-motile sperm cells fromdetritus. Further, the improved optical system 160 allows for a markedreduction in the proportion of tracking errors at a given sperm cellconcentration. The improved optical system 160 also allows for a moreaccurate analysis at higher values of sperm cell concentrations.

The improved optical system 160 essentially comprises a single LEDillumination source 162 combined with a small source aperture 164 formedin a suitable enclosure 166 for the source 162. By using a single LED asthe illumination source 162, the combination functions as a collimator.As such, it takes the place of both the collimating lens 56 and thecondensing lens 62 of the optical system 12 illustrated in FIG.1.Similar collimating and condensing lenses also are required inconventional phase contrast systems. See specifically the article "Someimprovements in the phase contrast microscope," by K. Yamamoto et al,Journal of Microscope, Vol. 129, Pt.1, January 1983, pp. 49-62, at p.50. See also, F. Zernike, "Das Phasenkontrastverfahren bei dermikroskopischen Beobachtung," Physik, Z. XXXVI, 1935; and F. Zernike,"Phase Contrast, A New Method for the Microscopic Observation ofTransparent Objects," Physica IX, No. 7, July 1942, pp. 686-698.Preferably, the single LED is a super-bright LED with a high level ofintensity, as represented by an LED, Type No. H-2000, operable at a peakwavelength of 660 nm, with a half-angle spread of 5° , as made by theStanley Electric Co., Ltd. of Japan. Other equally or more powerfulLED's; and other, shorter wavelengths, also can be employed..

Unlike both of the system of FIG.1 herein and the phase contrast systememployed in microscopes, in the improved optical system 160 of thepresent invention, all of the illumination emanating as beam 168 fromthe single LED source 162, as collimated by the precisely positionedsmall source aperture 164, is directed at the specimen contained withinthe specimen holder 50 supported on member 110. A part 170 of the beam168 represents light transmitted through the specimen, while a part 172is scattered by the specimen. Thus, both the directly transmitted light170 as well as the light scattered 172 by the specimen are directed by afirst reflecting element 174 at an imaging lens 176. The imaging lens176, in turn, focuses both parts 170 and 172 of the beam 168 onto thelight sensitive cells 78 of the light sensitive device 76 of theradiation sensing means 18, with the aid of a second reflecting element178. Both parts 170 and 172 of the beam 168 recombine upon reaching thedevice 76 to form the image at the screen 22a, see FIG. 1. The resultantimage is now presented in the bright field illumination mode, where thesperm cells appear as dark images on a relatively bright background.

The improved motility scanner according to FIG. 9 also can be operatedin a dark field illumination mode by incorporating an electronic imagereversal means 180 in the radiation sensing means 18, preferably in thesignal processing means 20. The image is inverted before analysis. Withthe aid of the electronic image reversal means 180, the image iselectronically reversed, before being analysed by the microprocessor 82,into an image in which the sperm cells appear as bright images on arelatively dark background.

The improved motility scanner according to FIG. 9 also can be operatedas a negative phase contrast system, resulting in heightened imageresolution. Phase contrast is characterized by bright, boldillumination, but gives a dark field image. Phase retardation of thebeam 168 is introduced into the system 160 by providing a speciallydesigned plate 182 at the plane conjugate to the plane of the smallsource aperture 164 of the LED illumination source 162. At thisconjugate plane, an image of the source aperture 164 is formed, thediameter of which image is the diameter of the source aperture 164multiplied by the magnification ratio of the optical system 160. Theplate 182 is designed to be moved between a first operative positionwithin the beam 168, illustrated in solid lines, to a second operativeposition, shown in phantom lines, outside of the beam 168, by anappropriately designed holder 184. Preferably, the motion is a rotatingone, swinging the plate 182 in or out of the beam 168, as desired by theuser. The plate 182 is provided with a centrally disposed coating 186 ofa retarding member 188 and/or an attenuating member 190, as more fullydescribed below with reference to FIGS. 10-13. As will be observed inFIG. 9, the part 170 of the beam 168 representing the directlytransmitted light travels through this centrally disposed coating 186.On the other hand, the part 172 of the beam 168 representing the lightscattered by the specimen in the specimen holder 50, for the most part,travels through the plate 182 outside of the centrally disposed coating186, the significance of which will be more fully described below.

Preferably, the plate 182 comprises a disk formed of an optical glasswindow, with a preferred diameter (D) of about 25 mm and a preferredthickness (T) of about 3 mm, see FIG. 10. The centrally disposed coating186 preferably is provided thereon by evaporation, using a speciallydesigned and constructed mask 192, observe FIG. 13. The mask 192 is sodesigned and constructed as to securely hold and mask the plate 182during the evaporation process, except for a central spot 194 with adiameter (d), the desired diameter for the coating 186. With a preferreddiameter for the coating 186 of about 0.115 inch and a magnificationratio of 2.875 for the optical system 160, a small source aperture 164of about 0.04 inch diameter is called for. It is understood that varyingthe size of one will affect the size of the remaining two variables,according to the relation expressed above.

As stated, the coating 186 comprises either the phase retarding member188, illustrated in FIG. 10, the attenuating member 190 illustrated inFIG. 11, or a combination of the retarding member 188 onto which theattenuating member 190 is deposited, preferably also by evaporation.Preferably, the retarding member 188 is formed with a thickness of about120 nanometers and of a group including magnesium fluoride and titaniumdioxide. Preferably, the attenuating member 190 also is formed from anickel-base alloy characterized by high temperature oxidationresistance, such as Inconel alloy 600. Preferably, the attenuatingmember 190 provides an attenuation of the part 170 of the beam 168transmitted therethrough of about 97%, transmitting only the remainderof about 3% of this part 170 of the beam 168.

With the plate 182 positioned by the holder 184 into the beam 168 of theimaging lens 176, the phase retarding member 188 of the coating 186 willretard the phase of the entire part 170, representing the directlytransmitted light, of the beam 168 by one quarter wavelength. The part172 of the beam 168, representing the light scattered by the specimenhowever, travels through the plate 182 for the most part outside of thereach of the phase retarding member 188 and is, therefore, not retarded.As a consequence, a sharp image is created in which even the tails ofthe sperm cells are clearly visible. And if the coating 186 alsoincludes the attenuating member 190 superimposed on the phase retardingmember 188, the contrast of the phase-contrast image is furtherincreased, making analysis of the image of the sample specimen even moreaccurate. For here again, the part 172 of the beam 168 representing thelight scattered by the specimen, in traveling through the plate 182 forthe most part outside of the coating 186, is thus neither attenuated norretarded relative to the directly transmitted part 170 of the beam 168.As mentioned, both parts 170 and 172 of the beam 168 recombine at thelight sensitive device 76 of the radiation sensing means 18 to form theimage projected onto the screen 22a of the motility scanner of theinvention. The result is an image with a sharply improved opticalresolution.

The improved optical system 160 further excels in being very stableagainst dislocation of the component parts during transport orinstallation. This is so since the system 160 is relatively tolerant ofline-up errors.

Effecting a selection among the three modes of operation (bright field,dark field, and negative phase contrast) is easily done by manipulatingone of the controls of the control means 24 located on the front panel36 of the instrument. The phase contrast plate holder 184 can also bemoved manually into or out of the beam.

The Preferred Process of the Invention

The preferred process of employing the motility detector 10 of theinvention for characterizing the motion of sperm, bacteria, particlessuspended in flowing fluids and the like will be illustrated withspecific reference to equine semen, specifically that of stallions.

Note however that this does not preclude the use of a Makler chamber orspecimen holder for more concentrated specimens. In using thephase-contrast optical system described in the embodiment of FIGS. 9-12,a Makler chamber (or Petroff-Hauser chamber) is indicated. This isbecause the depth of focus is greatly reduced in the use ofphase-contrast optics, and therefore the specimen should be maintainedwithin a 20 micron thickness. It should be pointed out, however, thatphase contrast can still be used with the specimen holding chamber 100described here. But the image of entire volume of chamber 100 cannot befocused simultaneously with phase-contrast optics onto the sensitivemeans 78. It is only possible to focus the cells swimming along theupper or lower walls of the chamber 100 when phase contrast optics isused.

A specimen sample, i.e., a drop of semen obtained from a stallion, ispreferably first diluted with a suitable diluent and in a predeterminedratio. A suitable diluent is Hepes transparent extender or Kenney's(powdered milk and glucose) equine extender. The predetermined ratio ofdiluent to sample for stallions is in the range of about 50:1 to about250:1, and preferably is about 250:1. The diluted sample is drawn up bycapillary action into the specimen holding channel 100 of a specimenholder 50, one end of which is immersed below the surface of the dilutedsemen. The specimen holder 50 containing the diluted sample specimen isthen placed on the heated specimen support 16, which is then moved backthrough the slot 52 into the motility scanner 10. As mentioned, thedimensions of the specimen holder 50 are such that it will slide withinthe channel guide 114 so that a portion of the specimen holder 50 willlie over the window 112 formed in the flat member 110, observe FIG. 3.

With the motility scanner 10 having been activated by turning the powerswitch 88 on, the flat member 110, preferably made of a thermallyconductive material, such as aluminum, is maintained at a temperature ofabout 37° C. by an embedded resistor 116, through which an electriccurrent is passed. A thermal sensor 118 also is embedded in the member110 and is used to control the current passing through the resistor 116,and thereby its temperature. The temperature can be set using thecontrol means 24. Member 110 is in turn maintaining the diluted samplewithin the specimen holder 50 at about the same temperature, i.e., aboutor slightly above normal body temperature. Due to this temperature andthe dilution of the sample in the specified ratio, a milieu for theanalyzed sample is being maintained throughout the process of theinvention, which milieu most closely resembles that environment throughwhich the sperm migrate normally and naturally. By depressing one of thecontrol buttons of the control means 24, the source of illumination 14and the signal processing means 20 are actuated and analysis of thespecimen sample is effected. Focussing means 120 is provided to vary thedistance between the imaging lens 64 and the specimen holder 50,producing thereby a sharp image at the image plane.

Since the effective depth of field of the imaging lens 64 at its objectplane, precisely where the specimen holder 50 is disposed, exceeds thedepth 102 of the specimen holder 50, the entire specimen depth issharply imaged on the light-sensitive cells 78 of the charge coupleddevice 76. Further, since the imaging lens 64 is designed to maximizethe modulation transfer function across a wide angular field, a highedge resolution of sperm in the analyzed sample is obtained. Spermwithin the sample specimen now appear as bright objects on a blackbackground. Successive images produced on the light sensitive cells 78,being representative of the sperm moving through the diluted sample, arethen transmitted to the frame grabber board typically at about fiveframes per second, although higher or lower rates of image acquisitionalso can be used. These transmitted images are now stored in the RAMthereof. After a plurality of images have been so stored in the RAM,namely at least about 5 and preferably about 20 to 30 stored images, theimage taking process ceases and analysis of the stored images commences.

A frame subtraction procedure first is employed which removes allstationary objects, caused by the presence of dirt or the like either inthe optical system 12 or within the specimen sample, from the process sothat only the moving parts, e.g., liver sperm, of the specimen sampleare dealt with by the system. A background frame is constructed from atleast one, typically three, of the recorded video frames, by comparingthese video frames and retaining, pixel by pixel, the minimum of thethree pixel values. The background frame constructed in this way issubtracted pixel by pixel, from each of the recorded vodeo frames. Theresulting video images contain only the non-static component of theoriginal images.

Following the removal of stationary objects from adversely affecting theprocess of motion characterization, analysis of the transmitted framesis then effected in and by the coded device 84, specifically by theplurality of ROM chips contained in the device 84. These ROM chips havepreviously been pre-programmed with instructions to analyse thetransferred images from the light-sensitive cells 78 of the device 76and to derive sperm velocity and linearity distribution functionstherefrom. The motion characterization process includes analysis of thesperm tracks in which the velocity along the sperm track and thevelocity between track end points are calculated, and the tracklinearity is derived as the ratio of the distance between the startingand the end points of the tracks to the total track length. Mean valuesof velocity and linearity are then computed and these averages, as wellas the distributions of number of sperm versus velocity and linearityare displayed on the screen 22a and, if desired, printed on a hard copyby the printer 22b. Alternately, the tracks may be plotted on a plotterperipheral device. A typical screen display and/or information appearingon a copy from the printer 22b is illustrated in Example I.

    ______________________________________                                        Example I                                                                     ______________________________________                                        DATA SUMMARY:      (time and date)                                            ______________________________________                                        TOTAL OBJECTS      50     million/ml. (185)                                   MOTILE OBJECTS     23     million/ml. (88)                                    MOTILE/TOTAL       47.6   percent                                             AVERAGE VELOCITY   32.5   microns/second                                      AVERAGE P-VELOCITY 24.1   microns/second                                      AVERAGE PROGRESS   73.4   percent                                             ______________________________________                                        PROGRESSIVITY DISTRIBUTION (PERCENT)                                          00-10  0                                                                      10-20  0                                                                      20-30  ****** 2                                                               30-40  ***************** 8                                                    40-50  ******** 3                                                             50-60  **************************** 12                                        60-70  ********************************************* 21                       70-80  ********************************************* 21                       80-90  ************************************* 16                                90-100                                                                              *************** 5                                                      VELOCITY DISTRUBITION (MICRONS/SEC)                                           00-10  ** 1                                                                   10-20  *************** 8                                                      20-30  ***************************************** 27                           30-40  ********************************************* 29                       40-50  ********************************* 19                                   50-60  ******* 3                                                              60-70  ** 1                                                                   70-80  0                                                                      80-90  0                                                                       90-100                                                                              0                                                                      ______________________________________                                    

Computer counting of sperm tracks then yields an accurate determinationof live sperm density, since the total volume of specimen examined isknown exactly from the optical magnification and the internal wallseparation of the specimen holder 50. In order to estimate the totalsperm density, the dead sperm cells are identified by selecting allbackground (non moving) objects whose optical brightness and lineardimensions fall within certain limits. These limits are defined asmultiples of the mean values determined for all the moving cells. Inthis way, use of an absolute illumination calibration can be avoided.The motility is then determined as the ratio of the number of mobileobjects to the total number of objects falling within the above limitsin the field.

Utilization of the motility scanner 10, together with the specimenholder 50, thus allows for accurate motion measurements of particlessuspended in a fluid because:

(a) the particles enjoy complete freedom of motion without beingconstricted by walls;

(b) the design of the specimen holder 50 allows ample time for effectingthe motion characterization of the specimen sample before any danger ofthe sample drying out;

(c) the optical system 12 allows for all of the spermatozoa or othermoving particles contained within the specimen sample within thespecimen holder 50 to be kept in clear focus throughout their movementwithin the depth 102 of the specimen holding channel 100;

(d) the known wall spacing of the specimen holder 50 allows exactdetermination of the volume, hence the density, of the sample; and

(e) the geometry of the specimen holder 50 minimizes problems of massfluid flows which might otherwise distort the data.

Thus there has been shown and described an improved motility scanner 10and method designed for characterizing the motion of sperm, bacteria,particles suspended in flowing fluids, Brownian motion and the like,which scanner 10 and method satisfy the objects and advantages set forthabove.

Since certain changes may be made in the present disclosure withoutdeparting from the scope of the present invention, it is intended thatall matter described in the foregoing specification or shown in theaccompanying drawings, be interpreted in an illustrative and not in alimiting sense.

What is claimed is:
 1. In a motility scanner of the kind forcharacterizing the motion of sperm, bacteria, particles in flowingfluids and the like, and comprising:(a) an optical system including animaging lens; (b) a source of illumination for said system; (c) aspecimen support including a specimen holder disposed in the objectplane of said imaging lens; (d) radiation sensing means coupled to saidsystem at the imaging plane of said imaging lens; (e) signal processingmeans for analyzing signals generated by said radiation sensing means;and (f) display means coupled to said signal processing means fordisplaying information characteristic of said motion;the improvementcomprising said source of illumination including a single LED and asmall source aperture designed to direct all of the illuminationemanating from said source onto said specimen holder, and a movableplate designed to be swung into the beam of said imaging lens at a planeconjugate to the plane of said small source aperture, said plateprovided with a centrally disposed retarding member and/or anattenuating member, with a part of said beam focussed by said imaginglens onto said retarding and/or attenuating member and with theremainder of said beam being focussed onto said plate surrounding saidretarding and/or attenuating member, said single LED being one of highintensity, the improvement further including an electronic imagereversal means forming a component part of said signal processing means,and switching means coupled to said electronic image reversal means foractuating the same.
 2. In the motility scanner of claim 1 wherein thediameter of said centrally disposed retarding and/or attenuating memberequals the diameter of said small source aperture multiplied by themagnification ratio of said optical system, and further including meansfor rotatably moving said movable plate from one operative position inthe beam of said imaging lens to a second operative position outsidesaid beam thereof.
 3. In the motility scanner of claim 1 wherein saidmovable plate is made from glass and having a diameter of about 25 mmand a thickness of about 3 mm, and wherein said centrally disposedretarding member is formed of one of a group consisting of magnesiumfluoride and titanium dioxide, and with a thickness of 120 nanometersand a diameter of about 0.115 inch.
 4. In the motility scanner of claim1 wherein said centrally disposed attenuating member comprises anabsorbing layer formed of a nickel-base alloy characterized by hightemperature oxidation resistance, and wherein said absorbing layereffects about 97% attenuation of that part of said imaging lens beamwhich is focussed thereon.
 5. In the motility scanner of claim 3 whereinsaid centrally disposed retarding and attenuating members are coated onsaid movable plate, wherein said coating of said centrally disposedretarding and attenuating members is effected by evaporation onto saidglass plate, and wherein said evaporation of said glass plate iseffected via a mask designed both to mask said glass plate and to holdit in place during said evaporation process.
 6. In the motility scannerof claim 5 wherein said retarding member is evaporated onto said glassplate first and said attenuating member is evaporated onto saidretarding member.
 7. In the motility scanner of claim 1 wherein theoperative use of said movable plate in conjunction with said electronicimage reversal means endows said motility scanner to be employed in oneof three operative modes: (a) with bright field illumination; (b) withdark field illumination; and (c) with phase contrast illumination, andwherein said small source aperture has a diameter of about 0.04 inch andwherein said magnification ratio of said optical system is about 2.875.8. In the motility scanner of claim 1 wherein said optical systemfurther includes at least one reflecting element disposed between saidsingle LED and small source aperture and said imaging lens.